The purpose of this work is to define the immunological basis that mediates protective immunity against Chlamydia trachomatis infection of the female genital tract. The long-term goal is to then use this information to develop a safe and efficacious vaccine against chlamydial caused sexually transmitted diseases (STD. The project involves the study of immunity in a murine model of chlamydial infection of the female genital tract. The goal of the work is to identify mechanisms of protective immunity and protective antigens, information that then can used to formulate novel vaccines to test in this pre-clinical model. The long-range goal is to move the most promising of these vaccines to human clinical trials to assess their safety and efficacy in preventing chlamydial STDs. Past studies from this laboratory using gene knock out mice, adoptive transfer of immune T cells, and in vivo depletion of T cell subsets strongly implicate CD4+ Th1 cell mediated immunity as the major protective arm of the immune response against chlamydial genital infection. Conversely, CD8+ T cells, gamma/delta T cells, and antibodies play only a limited role in mediating protective immunity. We have continued studies along these lines to further define effector function(s) of CD4+ Th1 mediated immunity and lymphocyte homing to the genital mucosal. Our findings support a role for IFN-gamma but not TNF-alpha, iNOS, or Fas mediated apoptopic killing in protective anti-chlamydial mediated T cell immunity. The effector mechanims by which IFN-gamma functions is through the induction of indoleamine 2,3-dioxygnease (IDO) and subsequent starvation of chlamydiae by the deprivation host tryptophan. Thus, vaccines capable of generating a chlamydial specific type 1 CD4+ immune response at the genital mucosae are likely to elicit protective immunity. Lymphoctye homing studies implicate both systemic and mucosal integrins and their cognate receptors in lymphocyte homing to the genital mucosa. To date, vaccination using conventional approaches such as targeted chlamydial recombinant proteins or DNA encoding proteins delivered either systemically or locally with and without Th1 promoting adjuvants have failed in eliciting a protective Th1 immunity at the genital mucosae. Infection of the genital tract or immunization with ex vivo pulsed dendritic cells (DC? are the only highly efficacious ways of generating protective immunity against chlamydial genital re-challenge. Our conclusions are that the antigenic complexity of the chlamydiae, its complex life cycle, and tropism for mucosal epithelial cells constitute overwhelming challenges for the generation of a conventional vaccine. Consequently, we are currently focusing on the generation of live-attenuated vaccine strains. This is being accomplished by the clonal selection of variants that are incapable of synthesizing tryptophan synthase, a key enzyme in the ability of the pathogen to persist in epithelial cells in the presence host defense. We are selecting attenuated trp-/- strains by growth in trp deficient medium supplemented with 5 fluoroindole (5-FI). Organisms with mutations in the tryptophane synthase gene (trpRBA) will not incorporate 5-FI and will survive its lethal effect. 5-FI resistant clones will be characterized biologically for sensitivity to IFN-gamma in vitro, indole rescuablility, and trpRBA sequence analysis. Clones whose growth is highly sensitive to IFN-gamma in vitro and that have mutations in the trpRBA genes will be tested in in vivo models of infection for attenuated growth and pathological properties. Strains demonstrating attenuated in vitro growth for the murine female genital tract will then be tested for their ability to induce protective immunity against challenge infections with virulent chlamydiae. These studies should yield information important to the development of a highly efficacious and safe vaccine capable of preventing or controlling chlamydial caused STDs in humans.